Synergistic effects of ε-poly-l-lysine and lysozyme against Pseudomonas aeruginosa and Listeria monocytogenes biofilms on beef and food contact surfaces.

This study investigated the synergistic effects of ε-poly- L -lysine (ε-PL) and lysozyme against P. aeruginosa and L. monocytogenes biofilms. Single-culture biofilms of two bacteria were formed on silicone rubber (SR), stainless steel (SS), and beef surfaces and then treated with lysozyme (0.05-5 mg/mL) and ε-PL at minimum inhibitory concentrations (MICs) of 1 to 4 separately or in combination. On the SR surface, P. aeruginosa biofilm was reduced by 1.4 and 1.9 log CFU/cm2 within 2 h when treated with lysozyme (5 mg/mL) and ε-PL (4 MIC), respectively, but this reduction increased significantly to 4.1 log CFU/cm2 (P < 0.05) with the combined treatment. On beef surface, P. aeruginosa and L. monocytogenes biofilm was reduced by 4.2-5.0, and 3.3-4.2 log CFU/g when lysozyme was combined with 1, 2, and 4 MIC of ε-PL at 25 °C, respectively. Compared to 5 mg/mL lysozyme alone, the combined treatment with 1, 2, and 4 MIC of ε-PL on beef surface achieved additional reduction against P. aeruginosa biofilm of 0.5, 0.8, and 0.7 log CFU/g, respectively, at 25 °C. In addition, 0.25 mg/mL lysozyme and 0.5 MIC of ε-PL significantly (P < 0.05) suppressed the quorum-sensing (agrA) and virulence-associated (hlyA and prfA) genes of L. monocytogenes.

PEG-functionalized aliphatic polycarbonate brushes with self-polishing dynamic antifouling properties.

Hydrophilic antifouling polymers provide excellent antifouling effects under usual short-term use conditions, but the long-term accumulation of contaminants causes them to lose their antifouling properties. To overcome this drawback, surface-initiated ring-opening graft polymerization (SI-ROP) was performed on the surface of the material by applying the cyclic carbide monomer 4'-(fluorosulfonyl)benzyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (FMC), which contains a sulfonylfluoride group on the side chain, followed by a "sulfur(IV)-fluorine exchange" (SuFEx) post click modification reaction to link the hydrophilic polyethylene glycol (PEG) to the polyFMC (PFMC) brush, and a novel antifouling strategy for self-polishing dynamic antifouling surfaces was developed. The experimental results showed that the antifouling surface could effectively prevent the adsorption of proteins such as bovine serum albumin (BSA, ∼96.4%), fibrinogen (Fg, ∼87.8%) and lysozyme (Lyz ∼69.4%) as well as the adhesion of microorganisms such as the bacteria Staphylococcus aureus (S. aureus) (∼87.5%) and HeLa cells (∼67.2%). Moreover, the enzymatically self-polished surface still has excellent antifouling properties. Therefore, this modification method has potential applications in the field of biosensors and novel antifouling materials.

Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

The molecular modification, expression, and the antibacterial effects studies of human lysozyme.

Human lysozyme (hLYZ) has attracted considerable research attention due to its natural and efficient antibacterial abilities and widespread uses. In this study, hLYZ was modified to enhance its enzyme activity and expressed in a Pichia pastoris expression system. A combination mutant HZM(2R-K)-N88D/V110S demonstrated the highest enzyme activity (6213 ± 164 U/mL) in shake flasks, which was 4.07-fold higher when compared with the original strain. Moreover, the recombinant P. pastoris was inducted in a 3 L bioreactor plus methanol/sorbitol co-feeding. After 120 h induction, the antibacterial activity of hLYZ reached 2.23 ± 0.12 × 105 U/mL, with the specific activity increasing to 1.89 × 105 U/mg, which is currently the highest specific activity obtained through recombinant expression of hLYZ. Also, hLYZ supernatants showed 2-fold inhibitory effects toward Staphylococcus aureus and Micrococcus lysodeikticus when compared with HZM(2R-K). Our research generated a hLYZ mutant with high antibacterial capabilities and provided a method for screening of high-quality enzymes.

Postbiotic, anti-inflammatory, and immunomodulatory effects of aqueous microbial lysozyme in broiler chickens.

Lysozymes, efficient alternative supplements to antibiotics, have several benefits in poultry production. In the present study, 120, one-day-old, Ross 308 broiler chickens of mixed sex, were allocated into 2 equal groups, lysozyme treated group (LTG) and lysozyme free group (LFG), to evaluate the efficacy of lysozyme (Lysonir®) usage via both drinking water (thrice) and spray (once). LTG had better (p = 0.042) FCR, and higher European production efficiency factor compared to LFG (p = 0.042). The intestinal integrity score of LTG was decreased (p = 0.242) compared to that of LFG; 0.2 vs. 0.7. Higher (p ≤ 0.001) intestinal Lactobacillus counts were detected in chickens of LTG. Decreased (p ≤ 0.001) IL-1ß and CXCL8 values were reported in LTG. The cellular immune modulation showed higher (p ≤ 0.001) opsonic activity (MΦ and phagocytic index) in LTG vs. LFG at 25 and 35 days. Also, higher (p ≤ 0.001) local, IgA, and humoral, HI titers, for both Newcastle, and avian influenza H5 viruses were found in LTG compared to LFG. In conclusion, microbial lysozyme could improve feed efficiency, intestinal integrity, Lactobacillus counts, anti-inflammatory, and immune responses in broiler chickens.

Exogenous aqueous and spray microbial lysozyme enhanced growth in commercial broiler chickensThe postbiotic effects of microbial lysozyme modulated intestinal integrity.Anti-inflammatory, as well as local, cellular, and humoral immune response were stimulated by lysozyme supplementation.

Oral administration of lysozyme protects against injury of ileum via modulating gut microbiota dysbiosis after severe traumatic brain injury.

Objective: The current study sought to clarify the role of lysozyme-regulated gut microbiota and explored the potential therapeutic effects of lysozyme on ileum injury induced by severe traumatic brain injury (sTBI) and bacterial pneumonia in vivo and in vitro experiments. Methods: Male 6-8-week-old specific pathogen-free (SPF) C57BL/6 mice were randomly divided into Normal group (N), Sham group (S), sTBI group (T), sTBI + or Lysozyme-treated group (L), Normal + Lysozyme group (NL) and Sham group + Lysozyme group (SL). At the day 7 after establishment of the model, mice were anesthetized and the samples were collected. The microbiota in lungs and fresh contents of the ileocecum were analyzed. Lungs and distal ileum were used to detect the degree of injury. The number of Paneth cells and the expression level of lysozyme were assessed. The bacterial translocation was determined. Intestinal organoids culture and co-coculture system was used to test whether lysozyme remodels the intestinal barrier through the gut microbiota. Results: After oral administration of lysozyme, the intestinal microbiota is rebalanced, the composition of lung microbiota is restored, and translocation of intestinal bacteria is mitigated. Lysozyme administration reinstates lysozyme expression in Paneth cells, thereby reducing intestinal permeability, pathological score, apoptosis rate, and inflammation levels. The gut microbiota, including Oscillospira, Ruminococcus, Alistipes, Butyricicoccus, and Lactobacillus, play a crucial role in regulating and improving intestinal barrier damage and modulating Paneth cells in lysozyme-treated mice. A co-culture system comprising intestinal organoids and brain-derived proteins (BP), which demonstrated that the BP effectively downregulated the expression of lysozyme in intestinal organoids. However, supplementation of lysozyme to this co-culture system failed to restore its expression in intestinal organoids. Conclusion: The present study unveiled a virtuous cycle whereby oral administration of lysozyme restores Paneth cell's function, mitigates intestinal injury and bacterial translocation through the remodeling of gut microbiota.

Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme.

This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 µg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 µg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 µg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 µg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Inhibitory effects of Bacillus velezensis ID-A01 supernatant against Streptococcus mutans.

BACKGROUND: Dental caries is a chronic oral disease caused by microbial infections, which result in erosion of the dental enamel and cause irreversible damage. Therefore, proper disease management techniques and the creation of an environment that prevents intraoral growth and biofilm formation of Streptococcus mutans in the early stages, are crucial to prevent the potential progression of dental plaque to disease. Here, we aimed to investigate antimicrobial and antibiofilm effects of the Bacillus velezensis ID-A01 supernatant (ID23029) against S. mutans, and its inhibitory effects on acidogenesis. RESULTS: A killing kinetics assay showed a peak lethality percentage of 94.5% after 6 h of exposure to ID23029. In sucrose-exposed conditions, ID23029 inhibited lactic acid formation, preventing the pH from falling below the threshold for enamel demineralization, and inhibited up to 96.6% of biofilm formation. This effect was maintained in the presence of lysozyme. Furthermore, ID23029 retained up to 92% lethality, even at an intraoral concentration at which lysozyme is ineffective against S. mutans. CONCLUSIONS: This study demonstrates the potential of the B. velezensis ID-A01 supernatant for the prevention and treatment of dental caries. Its eventual use in dental practice is encouraged, although further studies are required to confirm its beneficial effects.

Oral administration of terpenoids and phenol fraction of Padina gymnospora stimulates the nonspecific immune response and expression of immune genes, and protects the common carp (Cyprinus carpio) from experimental Aeromonas hydrophila infection.

Common carp (Cyprinus carpio), a valuable aquaculture species susceptible to various infections, requires effective immune enhancement strategies. This study investigates the immunomodulatory effects of orally administered terpenoids and phenol fraction (TPF) from Padina gymnospora in C. carpio, focusing on stimulation of nonspecific immune response, immune gene expression, and protection against experimental infection. P. gymnospora is a brown seaweed species known for its bioactive compounds and medicinal properties. TPF was extracted using the Harborne fractionation method, and the presence of terpenoids and phenol compounds was confirmed by qualitative analysis and high-performance thin layer chromatography (HPTLC). TPF was administered orally in different doses to carp. Nonspecific immune responses were evaluated by measuring cellular ROS, RNI, and peroxidase production. The expression of immune genes (lysozyme and interleukin-1ß) was assessed by reverse transcriptase PCR. Furthermore, the protective efficacy of TPF was determined by infecting carp with a virulent pathogen, Aeromonas hydrophila, and monitoring mortality rates and disease symptoms. The results demonstrate that oral TPF administration significantly enhances nonspecific immune responses, with increased ROS, RNI, and peroxidase production, indicating improved immune function. Expression levels of lysozyme and interleukin-1ß were upregulated, suggesting immune system activation. Moreover, TPF exhibited significant protection against experimental infection, with lower mortality rates compared to the control group. These findings highlight TPF's potential as an effective immunostimulatory agent, enhancing immune responses and providing infection protection in carp. In conclusion, oral TPF administration stimulates nonspecific immune responses, modulates immune gene expression, and confers protection against experimental infection in carp, displaying its potential for enhancing immune responses and disease resistance in aquaculture species, and contributing to sustainable fish health management.

[Effect of Fitofron on the persistence factors of opportunistic pathogens in urine isolated from patients with urinary tract infections].

AIM: To perform an experimental evaluation of the effect of Phytofron, used for the treatment of urinary tract infections, on the ability of opportunistic pathogens to inactivate innate immunity factors (lysozyme, pro- and anti-inflammatory cytokines) and form biofilms. MATERIALS AND METHODS: In vitro experiments were carried out on clinical isolates from urine of patients with pyelonephritis and cystitis: Escherichia coli, Staphylococcus aureus, S. haemolyticus, S. epidermidis, Enterococcus faecalis. The effect of Fitofron NPO FarmVILAR (Russia) on the anticytokine activity of bacteria against regulatory cytokines (IL4, IL6, IL8, TNF and IL17A) was determined by enzyme immunoassay, while anti-lysozyme trait and the ability to form biofilms was evaluated by the photometric method. RESULTS: The inhibitory effect of Fitofron on the ability of opportunistic microorganisms to inactivate innate immunity factors (lysozyme) and form biofilms, as well as the predominant inhibition of the studied cytokines, was experimentally established. CONCLUSION: Inhibition of the persistence factors of opportunistic pathogens by Fitofron, documented in vitro, can be considered as one of the possible mechanisms of its biological activity in vivo.

Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.

High starch diets attenuate the immune function of Micropterus salmoides immune organs by modulating Keap1/Nrf2 and MAPK signaling pathways.

Based on their good physiological functions and physical properties, carbohydrates are widely used in fish feed. However, excessive use of carbohydrates such as starch in fish feed may reduce the immunity of the fish and cause a series of health problems. In order to more clearly clarify the effects of different starch levels in feed on the immune organs of Micropterus salmoides, this study took the immune organs as the entry point and explored it from several perspectives, including differences in enzyme activity in plasma, changes in gene expression in immune organs, and resistance to pathogenic bacteria. The results showed that (1) high starch feed activates inflammatory responses in the spleen and head kidney through the MAPK signaling pathway. This leads to a decrease in the number of lymphocytes and weakens the resistance to pathogens; (2) high starch diet affects the antioxidant capacity of the trunk kidney by regulating the Keap1/Nrf2 pathway; (3) There was a strong correlation between gene expression patterns in the head kidney and lysozyme content in plasma. This implies that the high starch diet may regulate lysozyme production by affecting gene expression in the head kidney and further affect immune function. This study helps to reveal the interaction between starch and the immune system and provide scientific basis for the development of reasonable dietary recommendations and disease prevention.

Effect of microbial muramidase supplementation in diets formulated with different fiber profiles for broiler chickens raised under various coccidiosis management programs.

The objective of the present study was to determine the effects of muramidase (MUR) supplemented to diets formulated with different fiber sources (inert or fermentable) on the growth performance and intestinal parameters of broiler chickens raised under different coccidiosis management programs. A total of 2,208 male Ross 308 broilers were housed in 96 floor pens and distributed into a 2 × 3 × 2 factorial arrangement in a completely randomized block design with 2 sources of fiber (inert or fermentable fiber), 3 coccidiosis management programs (none, vaccine, or Salinomycin), and with or without supplementation of MUR at 35,000 LSU(F)/kg of diet. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were calculated for each feeding phase (d 0-14, d 14-28, d 28-36) and from d 0 to 36. On d 17 and d 31, samples were taken to analyze several parameters. The experimental data were analyzed with 3-way ANOVA considering the main effect of fiber source, coccidiosis program, inclusion of MUR, and their interactions using JMP 16.2. 16S rDNA sequencing of the ileal and cecal content was carried out to analyze the diversity, composition, and predictive function of the microbiota. From d 0 to 36, BWG increased (P = 0.05) by 2.5% in birds supplemented with Salinomycin (P = 0.04), and by 2.2% with MUR supplementation (P = 0.02). Salinomycin and MUR improved FCR (P < 0.0001) when compared to nonsupplemented birds. The supplementation of MUR, regardless of the coccidiosis management program, reduced the intestinal viscosity (P = 0.03). On d 31, the highest blood concentration of carotenoids was observed in chickens fed diets supplemented with Salinomycin. MUR led to significant changes in the diversity, composition, and predictive function of the ileal microbiota, mainly on d 31. The results observed herein further explain the positive effects of MUR on the growth performance of broiler chickens.

Lysozyme-immobilized bandage contact lens inhibits the growth and biofilm formation of common eye pathogens in vitro.

Bandage contact lenses have an increased affinity to accumulate tear film proteins and bacteria during wear. Among the wide variety of tear film proteins, lysozyme has attracted the most attention for several reasons, including the fact that it is found at a high concentration in the tear film, has exceptional antibacterial and antibiofilm properties, and its significant deposits onto contact lenses. This study aims to evaluate the effect of lysozyme on bacterial biofilm formation on bandage contact lenses. For this purpose, several methods, including microtiter plate test and Colony Forming Unit (CFU) assay have been used to determine antibacterial and antibiofilm characteristics of lysozyme against the two most frequent contact lens-induced bacterial ocular infections, Staphylococcus aureus, and Pseudomonas aeruginosa. The results of these assays demonstrate lysozyme potential to inhibit 57.9% and 80.7% of the growth of S. aureus and P. aeruginosa, respectively. In addition, biofilm formations of P. aeruginosa and S. aureus reduced by 38.3% and 62.7%, respectively due to the antibiofilm effect of lysozyme. SEM and AFM imaging were utilized to visualize lysozyme antibacterial activity and topography changes of the contact lens surface, respectively, in the presence/absence of lysozyme. The results indicated that lysozyme can efficiently attack both gram-positive and gram-negative bacteria and consequently lysozyme-functionalized bandage contact lenses can reduce the risk of ocular infection after eye surgery.

Tendon-inspired hybrid hydrogel based on polyvinyl alcohol and gallic acid-lysozyme for promoting wound closure and healing.

Noninvasive wound closure remains a challenge in the field of wound healing. In this study, we report the development of a cross-linked P-GL hydrogel constructed from polyvinyl alcohol (PVA) and GL (a hydrogel consisting of gallic acid and lysozyme) that effectively promotes wound closure and healing. The P-GL hydrogel exhibited a unique lamellar and tendon-like fibrous network structure, providing good thermo-sensitivity and tissue adhesiveness up to 60 MPa, as well as retaining autonomous self-healing and acid resistance capacities. In addition, the P-GL hydrogel exhibited sustained release characteristics lasting >100 h, excellent biocompatibility both in vitro and in vivo, as well as good antibacterial activity and mechanical properties. The in vivo full-thickness skin wounds model revealed the positive wound closure and healing therapeutic effects of the P-GL hydrogels were confirmed, showing a promising potential as a noninvasive wound closure and healing bio-adhesive hydrogel.

TLR4 activation by lysozyme induces pain without inflammation.

Mostly, pain has been studied in association with inflammation, until recent studies which indicate that during bacterial infections, pain mechanisms could be independent of the inflammation. Chronic pain can sustain long after the healing from the injury, even in the absence of any visible inflammation. However, the mechanism behind this is not known. We tested inflammation in lysozyme-injected mice foot paw. Interestingly, we observed no inflammation in mice foot paw. Yet, lysozyme injections induced pain in these mice. Lysozyme induces pain in a TLR4-dependent manner and TLR4 activation by its ligands such as LPS leads to inflammatory response. We compared the intracellular signaling of MyD88 and TRIF pathways upon TLR4 activation by lysozyme and LPS to understand the underlying mechanism behind the absence of an inflammatory response upon lysozyme treatment. We observed a TLR4 induced selective TRIF and not MyD88 pathway activation upon lysozyme treatment. This is unlike any other previously known endogenous TLR4 activators. A selective activation of TRIF pathway by lysozyme induces weak inflammatory cytokine response devoid of inflammation. However, lysozyme activates glutamate oxaloacetate transaminase-2 (GOT2) in neurons in a TRIF-dependent manner, resulting in enhanced glutamate response. We propose that this enhanced glutaminergic response could lead to neuronal activation resulting in pain sensation upon lysozyme injections. Collectively we identify that TLR4 activation by lysozyme can induce pain in absence of a significant inflammation. Also, unlike other known TLR4 endogenous activators, lysozyme does not activate MyD88 signaling. These findings uncover a mechanism of selective activation of TRIF pathway by TLR4. This selective TRIF activation induces pain with negligible inflammation, constituting a chronic pain homeostatic mechanism.

Promotion of degradative autophagy by 6-bromoindirubin-3'-oxime attenuates neuropathy.

Damage to the central or peripheral nervous system causes neuropathic pain. Endoplasmic reticulum (ER) stress plays a role in peripheral neuropathy. Increase in ER stress is seen in diabetic neuropathy. Inducers of ER stress also give rise to peripheral neuropathy. ER stress leads to the formation of autophagosome but as their degradation is also stalled during ER stress accumulation of autophagosomes is seen. Accumulation of autophagosomes has deleterious effects on cells. In the present study, we show that treatment with tunicamycin (TM) (ER stress inducer) in mice leads to peripheral neuropathy as assessed by Von Frey and Hot plate method. Administration of a promoter of autophagy viz. 6-bromoindirubin-3'-oxime (6-BIO) subsequent to ER stress induced by TM exhibits a decrease in peripheral neuropathy. 6-BIO was also effective in reducing diabetic peripheral neuropathy. To understand the type of autophagy activated, SH-SY5Y cells were treated with 6-BIO after TM treatment. Levels of cathepsin D (CTSD), a marker for degradative autophagy was higher in cells treated with 6-BIO after TM treatment compared to only TM-treated SH-SY5Y cells while levels of Rab8A,-a marker for secretory autophagy was reduced. Furthermore, in parallel during ER stress secretory, we noted increased levels of lysozyme in autophagosomes destined for secretion. Cells treated with 6-BIO showed reduction of lysozyme in secretory autophagosomes. This shows that 6-BIO increased degradative autophagy and reduced the secretory autophagy. 6-BIO also reduced the caspase-3 activity in 6-BIO-treated cells. Thus, 6-BIO reduced neuropathy in animals by activating degradative autophagy and reducing the secretory autophagy.

Heat-Denatured Lysozyme is a Novel Potential Non-alcoholic Disinfectant Against Respiratory Virus.

Respiratory diseases are significant recurrent threats to global public health. Since the 1918 Spanish flu pandemic, seasonal influenza viruses continue to cause epidemics around the world each year. More recently, the COVID-19 global pandemic conducted a public health crisis with more than 6 million deaths and it also severely affected the global economy. Due to the phenomenon that people get infection from objects carrying viruses, it has aroused people's attention to home disinfection. As there is no ideal existing common domestic disinfectant, new and safer antiviral disinfectants are urgently needed. Lysozyme is a natural antibacterial agent widespread in nature and widely used in healthcare and food industry because of is recognized safety. Recently, it has been shown that thermally denatured lysozyme has the ability to kill murine norovirus and hepatitis A virus. In our study, we also demonstrated that heat-denatured lysozyme (HDLz) had an antiviral effect against H1N1 influenza A virus, and we optimized its antiviral activities by testing different heating denaturation conditions, to generalize this property, using pseudotype virus neutralization assay, we found that HDLz can also inhibit the entry of H5N1, H5N6, and H7N1 avian influenza viruses as well as SARS-CoV and SARS-CoV-2 particles in cell with IC50 at the ng/mL range. Finally, using western blot analysis, we provide evidence that HDLz polymerization correlates with antiviral effect, which may be a precious possible quality control test. Altogether, our data support HDLz as a powerful anti-respiratory virus disinfectant as a sole or additive of current disinfectants to reduce concentration of toxic component.

Dietary muramidase improved growth performance, feed efficiency, breast meat yield, and welfare of turkeys from hatch to market.

Muramidase is an enzyme that hydrolyzes peptidoglycans of bacterial cell walls and improves performance of broilers in a dose-dependent manner. An experiment was conducted to evaluate muramidase supplementation, at a high or step-down dose, in turkeys from hatch to market. Male, B.U.T. 6 turkey poults were placed in 24 floor pens at 32 birds per pen. Poults were fed 1 of 3 diets from d 1 to 126 of age. There were 8 replicate pens per treatment. The treatments were a control (CTL) diet, the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 6 (BAL45), and the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 3 and decreased to 25,000 LSU(F)/kg from phase 4 to 6 (BAL45-25). Data were analyzed using SAS. The model included treatment and block and means were separated by Fisher LSD test. Birds fed BAL45 were heavier (P < 0.05) and had a greater (P < 0.05) average daily gain compared with birds fed the CTL from hatch to d 126 of age. Birds fed BAL45-25 had a final BW and average daily gain intermediate to or equivalent to birds fed BAL45 at the same phases. Feed conversion ratio was improved (P < 0.05) in birds fed BAL45 compared with birds fed the CTL and intermediate in birds fed BAL45-25. Breast meat yield was greater (P < 0.05) in turkeys fed muramidase, regardless of dose, compared with birds fed the CTL. There was no effect of treatment on muramic acid content in the jejunum digesta or litter scores. The frequency of pododermatitis score 1 was greater (P < 0.05) and score 2 was lower (P < 0.05) in birds fed muramidase, regardless of dose, compared with birds fed the CTL diet. In conclusion, muramidase supplementation improved performance, breast meat yield, feed efficiency and some markers of welfare, proportional to the dose in the diets.